Mutations in the N-terminal kinase-like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis
Mutations in the N-terminal kinase-like domain of the repressor of photomorphogenesis SPA1 severely impair SPA1 function but not light responsiveness in Arabidopsis
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DOI：
10.1111/tpj.13241
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发表时间：
2016-10-01
期刊：
影响因子：
7.2
通讯作者：
Hoecker, Ute
中科院分区：
文献类型：
--
作者：
Holtkotte, Xu;Dieterle, Stefan;Hoecker, Ute
COP1/SPA复合体是一种E3泛素连接酶，是暗生长植物光形态发生的关键抑制因子。虽然COP1和四种SPA蛋白都含有卷曲线圈和wd重复结构域，但SPA蛋白与COP1的不同之处在于携带COP1中不存在的n端激酶样结构域。在这里，我们分析了SPA1在缺乏任何其他spa蛋白的spa四重突变背景下表达时n端缺失和错义突变的影响。SPA1大n端缺失严重损害了转基因植株的SPA1活性，影响了幼苗黄化、叶片扩张和开花时间。该DN SPA1蛋白在体外和体内对COP1的亲和力明显降低，表明其n端参与了COP1/SPA复合物的形成。仅删除激酶样结构域高度保守的95个氨基酸不会严重影响SPA1的功能，也不会与COP1或隐色素相互作用。相反，这部分激酶样结构域的错义突变严重破坏了SPA1的功能，这表明这些突变对SPA1活性有重要的负面影响。因此，我们假设激酶样结构域的序列在进化过程中被保守，因为它携带了对SPA1在黑暗中的活性很重要的结构信息。SPA1的n端对幼苗的光响应并不是必需的，这表明在SPA1 n端结构域缺失的情况下，光感受器可以抑制COP1/SPA复合物。总之，这些结果揭示了SPA1 n端在抑制光形态发生中的重要而复杂的作用。
The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark-grown plants. While both COP1 and the four SPA proteins contain coiled-coil and WD-repeat domains, SPA proteins differ from COP1 in carrying an N-terminal kinase-like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N-terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N-terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This DN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N-terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase-like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase-like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase-like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N-terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N-terminal domain. Together, these results uncover an important, but complex role of the SPA1 N-terminus in the suppression of photomorphogenesis.